PROPARGYLETHOXYAMINO NUCLEOTIDES
Propargylethoxyamino nucleosides are disclosed having the structure wherein R 1 and R 2 are —H lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W 1 is —H or —H; W 2 is —H or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W 3 is —PO 4 , —P 2 O 7 , —P 3 O 10 , phosphate analog, or —OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.
1 . A nucleoside compound having the structure:
wherein:
R 1 and R 2 taken separately are selected from the group consisting of —H, lower alkyl protecting group, and label;
B is a 7-deazapurine, purine, or pyrimidine nucleoside base;
wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N 9 -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N 1 -position of the pyrimidine; and
wherein when B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;
W 1 is selected from the group consisting of —H and —OH;
W 2 is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and
W 3 is selected from the group consisting of —PO 4 , —PO 2 O 7 , —P 3 O 10 , phosphate analog, and —OH.
2 . The nucleoside compound of claim 1 wherein one of R 1 and R 2 is label.
3 . The nucleoside compound of claim 2 wherein the label is a fluorescein-type dye.
4 . The nucleoside compound of claim 2 wherein the label is a rhodamine-type dye.
5 . The nucleoside compound of claim 1 wherein W 1 is —H and W 2 is —OH or a moiety, and W 3 is —P 3 O 10 .
6 . The nucleoside compound of claim 1 wherein W 1 is —H, W 2 is —OH or a moiety, and W 3 is —P 3 O 10 .
7 . The nucleoside compound of claim 1 wherein W 2 is —H or a moiety selected from the group consisting of —H, azido, amino, fluro, and methoxy.
8 . The nucleoside of claim 1 wherein B is selected from the group consisting of uracil, cytosine, 7-deazaadenine, and 7-deazaguanosine.
9 . A method for performing a primer extension reaction comprising the steps of:
providing a template nucleic acid;
annealing an oligonucleotide primer to a portion of the template nucleic acid; and
adding primer-extension reagents to the primer-template hybrid for extending the primer, the primer extension reagents including a nucleoside compound having the structure:
wherein:
R 1 and R 2 taken separately are selected from the group consisting of —H, lower alkyl, protecting group, and label;
B is a 7-deazapurine, purine, or pyrimidine nucleoside base;
wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N 9 -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N 1 -position of the pyrimidine; and
wherein if B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, if B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and if B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;
W 1 is selected from the group consisting of —H and —OH;
W 2 is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and
W 3 is selected from the group consisting of —PO 4 , —P 2 O 7 , —P 3 O 10 , phosphate analog, and —H.
9 . The method of claim 9 wherein one of R 1 and R 2 is label and the other is —H.